(i) Field of the Invention
The present invention relates to a gene encoding an enzyme having a flavin reducing activity and a nitroreductase activity, the enzyme produced therefrom, a recombinant vector containing the gene and bacteria containing the recombinant vector.
(ii) Description of the Related Art
A bacterial luciferase derived from luminous bacteria is used to produce oxidized flavin adenine mononucleotide ( hereinafter referred to as "oxidized FMN") and a long-chain carboxylic acid in the presence of a long-chain aliphatic aldehyde, oxygen and a reduced flavin adenine mononucleotide (hereinafter referred to as "FMNH.sub.2 ") as a luminescent substrate, and in this case, the bacterial luciferase catalyzes a reaction in which blue light is emitted. FMNH.sub.2 which is a substrate can be obtained from a reduced nicotinamide adenine dinucleotide: flavin mononucleotide (NADH:FMN) reductase and a reduced nicotinamide adenine dinucleotide phosphate: flavin mononucleotide (NADPH:FMN) reductase, and the long-chain aldehyde can be obtained from a fatty acid reductase complex.
In recent years, Spyrou et al. have isolated a flavin reductase gene from Escherichia coli and elucidated its primary structure, which is disclosed in Spyrou G., Haggard-Ljungquist E., Krook M., Jornvall H., Nilsson E. and Reichard P., J. Bacteriol, 173, p. 3673-3679 (1991).
Around us, there are many substances (mutagens) which damage chromosomal DNA, and during our lives we are exposed to these substances. Nitroarenes are members of one group of environmental mutagens, and they are contained in the exhaust gas of automobiles, the smoke of incinerators, the atmosphere of cities, the bottoms of rivers, the air in rooms where stoves are lighted, and the burnt portions of grilled chickens. Of nitroarenes having mutability and carcinogenicity, 2-nitrofluorene is well known.
A nitroarene itself does not react directly with DNA to damage the same, but a metabolite of the nitroarene gives rise to a mutation in DNA to damage the DNA. For example, it can be presumed that nitrofluorene is reduced to an N-hydroxy form in the cell of a microorganism by a nitroreductase and then activated by an o-acetyl transferase, to thus finally produce nitrenium ions which attack the DNA. Therefore, it can be considered that the reaction of the nitroreductase with 2-nitrofluorene is a rate determining step in the mutagenesis of DNA by 2-nitrofluorene.
Watanabe et al. have isolated a nitroreductase gene from Salmonella, which is disclosed in Watanabe M., Ishidate M, Jr and Nohmi T., Mutation Research, p. 216 211-220 (1989). Furthermore, its primary structure has been elucidated in Watanabe M., Ishidate M, Jr and Nohmi T., Nucleic Acid Research, 18, p. 1059 (1990).
As understood from the foregoing, the FMN reductase is essential to utilize the luminescent reaction of bacterial luciferase to the utmost. Therefore, the isolation of the FMN reductase gene permits preparing the enzyme in large quantities, and thus, an important object is the isolation of the gene encoding this enzyme.
Furthermore, the nitroreductase gene is useful to improve the detection sensitivity of the above-mentioned mutagen or carcinogen.
However, with regard to the isolation of the FMN reductase gene of luminous bacteria and the nitroreductase gene as well as the expression of them in Escherichia coli, no report has been made so far.